Tissue inhibitor of matrix metalloproteinases type 4 (TIMP-4), the newest member in the mammalian TIMP family of inhibitors of matrix metalloproteinases (MMPs), differs from the other three TIMPs by its restricted expression pattern. This suggests that TIMP-4 could play a role in tissue-specific regulation of extracellular matrix (ECM) turnover. To define this role, modulation of TIMP-4 production by overexpression, aberrant expression and inactivation of the Timp4 gene in transgenic mice should be performed. In preparation for such experiments we have cloned and characterized the murine Timp4 gene and determined the tissue distribution of its mRNA in mice. The gene spans 7.1 kb, consists of five exons and shares considerable homology with the other Timp genes. The gene is located on mouse chromosome 6 in an antisense orientation between exons 5 and 6 of the mouse synapsin 2 (Syn2) gene. A similar organization is common to all four human/mouse TIMP and SYN genes and to the single synapsin/Timp locus in Drosophila. The highest levels of TIMP-4 mRNA were seen in postnatal mouse heart, ovary and brain. Determination of the spatial expression pattern of TIMP-4 mRNA by in situ hybridization in the heart revealed a diffuse distribution in cardiac muscle cells. In the ovary, cyclic variation was observed in TIMP-4 mRNA levels. In situ hybridization demonstrated the strongest expression of TIMP-4 mRNA in the corpus luteum. The data suggest that TIMP-4 plays a role in the normal physiology of the heart and the ovary, most likely related to maintenance of the delicate balance between MMPs and TIMPs.